Engineering the Saccharomyces cerevisiae β-Oxidation Pathway to Increase Medium Chain Fatty Acid Production as Potential Biofuel

Abstract

Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain Δpox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the Δpox1 strain. The resulting Δpox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT) strain, the total fatty acid production of the strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio accumulate in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid-rich feedstock into high value fatty acid-derived products.

Urinary biomarker and treatment mechanism of Rhizoma Alismatis on hyperlipidemia

“…In comparison with control rats, hypoxanthine was obviously down‐regulated in diet‐induced hyperlipidemic rats, which was consistent with a previous study (Jiang et al, , b). Our previous study showed that 3‐methyluridine is a biomarker in rats with hyperlipidemia and 3‐methyluridine was restored after treatment with rhubarb (Chen et al, , b, c). In addition, 3‐methyluridine is associated with fat mass and obesity‐associated gene and RNA modification analyses uncovered that loss of fat mass and obesity gene affects the 3‐methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA (Berulava et al, ).…”

“…In this study, propionylcarnitine, methylhippuric acid, 4‐aminohippuric acid, hippuric acid and phenylacetylglycine were involved in fatty acid metabolism of RA‐treated hyperlipidemic rats. Compared with the control group, urinary hippuric acid and methylhippuric acid in diet‐induced hyperlipidemic group were higher, which was recognized as a possible sign of atherosclerosis (Zhang et al, , b; Chen et al, , b, c). However, after oral administration of RA, their abnormalities were restored to normal.…”

“…However, after oral administration of RA, their abnormalities were restored to normal. As a kind of fatty amide, methylhippuric acid is a secondary metabolite of fatty acids, which mainly undergoes β ‐oxidation in hepatic peroxisomes and mitochondria (Chen et al, , b, c). Increased methylhippuric acid corresponded to anomalous mitochondrial functions related to fatty acid β ‐oxidation, which can be a diagnostic indicator for such diseases.…”

“…Rhizoma Alismatis or Alisma orientale (RA), the dried rhizome of Alisma orientale Juzepzuk (Alismataceae), is usually used as an agent for diuresis and excreting dampness in Asia and Europe (Chen et al, ; Chen, Miao, Feng, Zhao, & Lin ; Chen, Zhang, & Chen, ; Feng et al, ). Additionally, RA is used to clinically treat hyperlipidemia in China.…”

Compartmentalized metabolic engineering for biochemical and biofuel production

“…Peroxisomes have recently been used to enhance the microbial production of biofuels . While many long chain organic acids can be produced by commonly used microorganisms such as Saccharomyces cerevisiae , medium chain organic acids are preferred for use as biofuels .…”